The goals of this research are to apply modern nuclear magnetic resonance techniques to chemical studies of protein structure and function. Certain well-defined protein systems are under investigation. It is hoped that the research will lead to improved methods for the resolution, assignment, and interpretation of NMR data as well as to solutions to questions concerning the protein systems themselves. We have recently succeeded in resolving 1H-NMR peaks corresponding to the histidine ring C(2)-protons of trypsin, trypsinogen, chymotrypsin, and chymotrypsinogen. The potential for observing spectroscopic signals from the catalytic histidine (His57) and from the histidine involved in the stabilization of the zymogen forms of these enzymes (His40 in the chymotrypsinogen numbering system) opens up a promising new research area. It now is possible to study the effects of a number of well-known chemical and enzymatic modifications of serine proteinases on the catalytic charge relay triad. It is possible as well to determine accurate pK' values for the 2 protonation steps affecting the charge relay system. In another series of experiments in collaboration with Professor M. Laskowski, Jr., at Purdue, we have introduced chemically synthesized amino acids enriched in certain sites with 13C into the reactive site peptide residue P1 of a natural serine proteinase inhibitor (soybean trypsin inhibitor, Kunitz). The 13C-NMR chemical shifts and relaxation times will be studied in the virgin and modified inhibitors and in complexes of the inhibitors with trypsin and chymotrypsin. These studies should provide information about the mechanism of interaction between the proteinase and inhibitor molecules. BIBLIOGRAPHIC REFERENCES: "The Charge-relay System of Serine Proteinases: Proton Magnetic Resonance Titration Studies of the Four Histidines of Porcine Trypsin," J.L. Markley and M.A. Porubcan, J. Mol. Biol. 102, 487 (1976). "Charge Relay of Serine Proteinases: NMR Studies of Free and Inhibited forms of Trypsin and Chymotrypsin," I.B. Ibanez, M.A. Porubcan, and J.L. Markley, Miami Winter Symposia, Volume 11, "Proteolysis and Physiological Regulation, D.W. Ribbons and K. Brew, Eds., Academic Press, New York, 1976.